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Abstract

Background

Activation of spinal cord glial cells such as microglia and astrocytes has been shown
to regulate chronic opioid-induced antinociceptive tolerance and hyperalgesia, due
to spinal up-regulation of the proinflammatory cytokines such as interleukin-1 beta
(IL-1β). Matrix metalloprotease-9 (MMP-9) has been implicated in IL-1β activation
in neuropathic pain. However, it is unclear whether acute opioid treatment can activate
glial cells in the peripheral nervous system. We examined acute morphine-induced activation
of satellite glial cells (SGCs) and up-regulation of IL-1β in dorsal root ganglia
(DRGs), and further investigated the involvement of MMP-9 in these opioid-induced
peripheral changes.

Our previous study has shown that matrix metalloproteinase 9 (MMP-9) plays an important
role in neuroinflammation and neuropathic pain development in part through the activation
(active cleavage) of IL-1β [20]. MMP-9 has also been shown to regulate the phenotype and proliferation of peripheral
[21] and central [20] glial cells. In a parallel study, we also showed that acute morphine induced a rapid
up-regulation of MMP-9 in primary sensory neurons in the dorsal root ganglia (DRGs),
masking morphine-induced analgesia (Liu et al., unpublished data). However, the molecular
and cellular mechanisms by which MMP-9 suppresses/shortens acute opioid analgesia
are unclear.

Satellite glial cells (SGCs) are peripheral glial cells and form a continuous layer
around primary sensory neurons within DRGs and trigeminal ganglia (TGs). SGCs have
been shown to regulate neuronal homeostasis and neurotransmission in DRGs and TGs
[22]. Increasing evidence suggests that SGCs exhibit marked morphological and biochemical
changes following peripheral nerve injury and inflammation [23-27]. Peripheral inflammation suppresses inward rectifying potassium currents and increases
IL-1β expression in SGCs of TG, leading to neuronal firing and trigeminal pain [28,29]. Chemokine signaling in DRG SGCs and neurons was shown to regulate chronic opioid-induced
hyperalgesia [30]. However, it is unclear whether and how SGCs are activated in DRGs following acute
opioid treatment. Our data demonstrated that a single subcutaneous morphine injection
could induce MMP-9 and opioid receptors-dependent activation of SGCs and IL-1β in
DRGs, highlighting the importance of peripheral neuronal-satellite glial interactions
for the control of acute opioid analgesia.

Results

Subcutaneous morphine induces GFAP expression in DRGs but not in spinal cords

The first question we addressed is whether acute morphine-induced acute analgesia
is associated with peripheral glial responses in DRGs. We used GFAP as a marker for
SGCs and performed immunohistochemistry to examine GFAP expression in DRGs, 2 h after
saline and morphine injection when morphine analgesia declines. In DRGs of saline-treated
control mice, only weak GFAP staining was observed (Figure 1A). Strikingly, the intensity of GFAP staining was markedly increased in DRGs of morphine-treated
mice (Figure 1B).

Figure 1.Subcutaneous morphine increases GFAP immunoreactivity in DRG SGCs. (A, B) GFAP immunoreactivity in DRGs from saline (A) or morphine (B, 10 mg/kg, s.c.) treated
mice at 2 h after the injections. Left panels are images with high contrast showing
all DRG cells in an optic field. Middle and right panels are low and high magnification
images of GFAP staining. Note there is a marked increase in GFAP staining after acute
morphine treatment. Scales, 50 μm.

Next, we performed qPCR analysis to quantify the GFAP mRNA levels in DRGs and spinal
cords at different times of morphine injection. Acute morphine increased GFAP mRNA
expression in DRGs [F(3,12) = 30.9, P < 0.0001, One-Way ANOVA], which peaked at 2 h (4.5 fold of control, P < 0.05, n = 4) and declined but still remained elevated at 3 h (2.6 fold of control,
P < 0.05, n = 4) (Figure 2A). In contrast, acute morphine did not change GFAP mRNA expression in spinal cord
dorsal horns at all the time points we examined (F(3,12) = 0.51, P = 0.682, One-Way ANOVA] (Figure 2B). Thus, it appears that acute morphine only causes reaction of peripheral glia (SGCs)
but not of central glia, since GFAP is a hallmark for astrocytes in the CNS.

In a parallel study, we demonstrated MMP-9 up-regulation in DRG neurons after acute
morphine treatment (Liu et al., unpublished data). MMP-9 could be secreted to extracellular
space to trigger the reaction of surrounding SGCs via neuronal-glial interaction.
Double staining of MMP-9 and GFAP and confocal microscopy examination revealed that
MMP-9 and GFAP-labeled structures were in very close proximity but not overlapped
(Figure 3D), providing a structural base for MMP-9-initiated neuronal-glial interaction in DRGs.

Subcutaneous morphine induces IL-1β activation in DRG SGCs via MMP-9

Chronic morphine but not acute morphine was shown to increase IL-1β levels in the
spinal cord [11]. We set out to test if acute morphine would induce IL-1β expression and activation
in DRGs. Low level of IL-1β expression was observed in DRGs of saline-treated wild-type
animals: only 5.4% DRG neurons were surrounded by IL-1β-expressing staining in the
control animals. However, this percentage increased to 22.7 in morphine-treated wild-type
mice (Figure 4A, B; P < 0.05, n = 6). Notably, this increase was abrogated in Mmp9-/- mice, and only 7.4% DRG neurons were surrounded by IL-1β-expressing satellite cells
(Figure 4A, B; P < 0.05, n = 6). However, acute morphine did not alter the expression of the IL-1β
mRNA (Figure 4C).

Next, we conducted Western blot analysis to check the activation of IL-1β in DRGs
after morphine treatment. Morphine produced no significant change of the 28 kDa band
of IL-1β, the non-mature form of IL-1β (Figure 4D). In contrast, morphine induced a significant increase of the 17 kDa band of IL-1β,
the active form of IL-1β, in DRGs of wild-type mice (Figure 4D, E, 1.4 fold of control, P < 0.05, n = 4). Notably, this increase of the active form of IL-1β was completely
abolished in Mmp9-/- mice (Figure 4D, E).

To determine the cellular localization of IL-1β in DRGs, we carried out triple staining
for GFAP, IL-1β, and DAPI (nuclear marker). We found a clear co-localization of GFAP
and IL1β in cytoplasm of SGCs of morphine-treated mice (Figure 5). Together, our results suggest that MMP-9 is required for the morphine-induced IL-1β
activation in SGCs.

To the best of our knowledge, this is the first report showing that acute morphine
could induce satellite glial activation in DRGs. We found significant increases in
both mRNA and protein levels of the satellite glial marker GFAP after a single subcutaneous
injection of morphine (Figures 1 and 2), indicating a peripheral glial reaction by acute morphine. In sharp contrast, previous
studies have focused on chronic morphine treatment and central glial activation. For
example, chronic morphine induced up-regulation of the microglial markers CD11b and
IBA1 and the astrocyte marker GFAP in the spinal cord [8,9,12,35,36]. In particular, chronic morphine activates p38 MAP kinase in spinal cord microglia
to promote morphine tolerance [10,36-38]. However, we failed to detect increases in the expression of spinal glial markers
such as the astrocyte marker GFAP (Figures 2 and 6) and the microglial marker IBA-1 (data not shown) after single subcutaneous and intrathecal
injections of morphine, suggesting that acute morphine primarily induces peripheral
glial responses in DRGs.

SGCs in the DRG are tightly associated with sensory neurons via gap junction; and
gap junction communication between SGCs and neurons is greatly enhanced in persistent
pain conditions [39,40]. Neuron-glial interactions in DRGs may involve purinergic signaling [41,42]. After nerve injury, chronic inflammation, and DRG compression, SGCs exhibit robust
up-regulation of GFAP [43-45]. Nerve injury-induced SGC reaction depends on neuronal activity and local inflammation
[23,46]. Our data showed that morphine-induced GFAP induction in SGCs required MMP-9 (Figure
3). As an extracellular matrix protein, MMP-9 could be released from DRG neurons following
morphine stimulation and retain in extracellular space between neurons and satellite
cells. MMP-9 may cleave extracellular matrix proteins to induce satellite glial reaction
and GFAP up-regulation (Figure 8). Of note MMP-9 immunostaining in neurons had very close proximity to GFAP immunostaining
in SGCs (Figure 3D). MMP-9 has been shown to regulate phenotypic remodeling and proliferation of peripheral
glial cells such as Schwann cells via signaling of IGF-1, ErbB receptors, and ERK
[21].

In particular, our data demonstrated that MMP-9 could drive IL-1β activation after
acute morphine. Acute morphine induced IL-1β activation in the DRG but not in the
spinal cord (Figures 4 and 6). Similarly, a previous study failed to show IL-1β increase either in lumbar spinal
cord or in CSF after acute morphine injection [11]. As previously reported [32,47,48], we observed IL-1β expression by DRG SGCs. However, we should not rule out the possibility
that IL-1β could also be expressed in neurons by testing different IL-1β antibodies
[32,49]. Primary sensory neurons particularly nociceptors express IL-1 receptors, and notably,
activation of these receptors by IL-1β can rapidly activate nociceptors to generate
action potentials and elicit pain hypersensitivity [50]. It appears that IL-1β increases the excitability of sensory neurons via increasing
sodium currents and decreasing potassium currents [48,50,51]. Importantly, intrathecal interleukin-1 receptor antagonist potentiates both intrathecal
and systemic morphine-induced analgesia [11,31]. Since IL-1β activation following acute morphine was primarily found in the DRG,
we postulate that intrathecal interleukin-1 receptor antagonist may also enhance opioid
analgesia by blocking IL-1β signaling in DRGs. This notion is strongly supported by
our siRNA experiment, showing that morphine analgesia was prolonged after IL-1β knockdown
in DRGs by selective siRNA treatment (Figure 7).

Importantly, Mmp9 deletion resulted in a complete loss of morphine-induced IL-1β activation in the DRG
(Figure 4). IL-1β is activated via cleavage from its precursor (≈ 31 kDa) and this activation
is not only mediated by caspase-1 (i.e. IL-1β converting enzyme) but also mediated
by other enzymes such as trypsin, elastase, collagenase, and cathepsin G, and MMP-9
[52-54]. MMP-9 can cause direct activation of IL-1β (generation of 17 kDa form) in a cell-free
system [53]. Notably, caspase-1-deficient mice can still produce bioactive IL-1β and mediate
IL-1β-dependent reactions under several pathological conditions [55]. Consistently, nerve injury-induced DRG-IL-1β activation is abolished in Mmp9-/- mice, whereas intrathecal MMP-9 increases IL-1β activation in DRGs [32]. However, we should not exclude the possibility that at very high concentrations
MMP-9 may also cause IL-1β degradation and inactivation via direct cleavage of IL-1β
at different sites [56].

In summary, opioids are still the most used analgesics for the treatment of moderate
to severe pain, despite their well-known side effects. Increasing evidence shows that
spinal cord glial cells and proinflammatory cytokines contribute to chronic opioid-induced
antinociceptive tolerance and hyperalgesia via neuronal-glial interactions in the
spinal cord [12,13]. We have demonstrated that acute morphine also induces peripheral satellite glial
cell (SGC) activation in the DRG, which is triggered by the protease MMP-9. Morphine-induced
MMP-9 up-regulation in DRG neurons could mask morphine analgesia via neuronal-glial
interactions. Specifically, MMP-9 caused IL-1β activation, leading to hyperexcitability
of primary sensory neurons to mask morphine analgesia (Figure 9). Thus, opioids not only produce excitatory effects in the spinal cord via postsynaptic
[57,58] and presynaptic mechanisms [59], they could further elicit excitatory effects at DRG level via MMP-9-triggered peripheral
neuronalglial interactions. Targeting peripheral neuronal-glial interactions in DRGs
may prolong the analgesic efficacy of opioids.

Methods

Animals

All experiments were performed in accordance with the guidelines of the National Institutes
of Health and the International Association for the Study of Pain. All animals were
used under Harvard Medical School Animal Care institutional guidelines. Adult male
mice (25-35 g) were used for behavioral and biochemical studies, including CD1 mice
(Charles River Laboratories, Wilmington, MA), Mmp9 konckout (Mmp9-/-) mice with FVB background, and FVB control wild-type mice. Mmp9 knockout (KO) mice and FVB wild-type mice were obtained from Jackson Laboratories
(The Jackson Laboratory, Bar Harbor, ME). The KO mice are viable, fertile, and maintained
in FVB background for more than 5 generations. Mice that are homozygous null for the
Mmp9 gene were used in this study. As previously described, they do not show any difference
in overall weight and behavior compared with wild-type mice [32]. Animals were housed in a 12 h light/dark room with access to food and water ad libitum.

Drugs and administration

We purchased morphine sulphate and remifentanil from Hospira (Lake Forest, IL) and
naloxone form Sigma (St. Louis, MO). Drugs were freshly prepared in saline and administered
subcutaneously in the lower back (10 mg/kg for morphine and naloxone) or intrathecally
(10 nmol for morphine and 1 nmol for remifentanil). The IL-1β (GCUCCGAGAUGAACAACAA)
and control siRNAs (GACUUCGCGGGACACAUGA) were purchased from Dharmacon (Dharmacon,
Inc., Chicago, IL). siRNA was dissolved in RNase-free water at the concentration of
1 μg/μl as stock solution, and mixed with polyethyleneimine (PEI, Fermentas Inc.,
Glen Burnie, MD), 10 min before injection, to increase cell membrane penetration and
reduce the degradation. PEI was dissolved in 5% glucose, and 1 μg of siRNA was mixed
with 0.18 μl of PEI. siRNA (3 μg) was intrathecally injected 24 and 2 h before the
morphine injection. Of note, agents with different chemical properties have been shown
to affect DRG cells via intrathecal route, including small molecules such as MAP kinase
and MMP-9 inhibitors [32,60] and large molecules such as growth factors and peptides [61,62], as well as antisense oligodeoxynucleotides [63,64] and siRNAs [33,65]. Intrathecal injection of p38 inhibitor can rapidly inhibit p38 activation in DRG
neurons within half hour [66]. Thus, intrathecal delivery of siRNA should affect cells both in DRGs and spinal
cords.

Behavioral test

All animals (n = 6 mice per group) were habituated to testing environment for at least
2 days before baseline testing. Morphine analgesia was evaluated by measuring the
tail-flick latencies in hot water [67]. Briefly, tail-flick test was performed by gently holding the mouse wrapped with
a terry towel and kept tail exposed. Then one third of the length of the tail was
immersed into the 52°C hot water, and the response latency was defined as removal
of the whole tail from the water. A maximum cut-off value of 10 seconds was set to
avoid thermal injury.

Immunohistochemistry

Two hours after morphine or vehicle (saline) injection, animals (n = 6 mice per group)
were terminally anesthetized with isoflurane and perfused through the ascending aorta
with PBS, followed by 4% paraformaldehyde with 1.5% picric acid in 0.16 M PB. After
the perfusion, DRGs (L4/L5) were removed and postfixed in the same fixative overnight.
DRG sections (12 μm) were cut in a cryostat and processed for immunofluorescence.
All the sections were blocked with 10% goat serum, and incubated over night at 4°C
with the primary antibodies against GFAP (mouse, 1:2000, Millipore, Billerica, MA),
MMP-9 (1:1000, rabbit, Millipore), or IL-1β (1:1000, rabbit, Millipore; 1:500, goat,
R&D System, Minneapolis, MN) with 5% goat serum. The sections were then incubated
for 1 h at room temperature with Cy3- or FITC-conjugated secondary antibody (1:400,
Jackson Immunolab, West Grove, PA) with 1% goat serum. DAPI (4',6-diamidino-2-phenylindole;
Sigma) staining was used to determine the cell nuclei. For double immunofluorescence,
sections were incubated with a mixture of polyclonal and monoclonal primary antibodies
followed by a mixture of FITC- and CY3-congugated secondary antibodies [68]. The stained sections were examined under a Nikon fluorescence microscopy, and images
were captured with a CCD camera (SPOT, Diagnostic Instruments). To obtain high-resolution
images, confocal images were captured from some DRG section with a Zeiss LSM 510 META
upright confocal microscope. All images were analyzed with NIH Image software or Adobe
Photoshop.

Western blot

Animals (n = 4-6 mice per group) were terminally anesthetized with isoflurane at 1,
2, and 3 h after morphine injection and transcardially perfused with PBS. DRGs (L4/L5)
and spinal cord segments (dorsal part of L4-L5) were rapidly removed and homogenized
in a lysis buffer containing a cocktail of protease inhibitors and phosphatase inhibitors
[69]. The protein concentrations were determined by BCA Protein Assay (Pierce Biotechnology,
Inc., Rockford, IL). Twenty micrograms of proteins were loaded for each lane and separated
on SDS-PAGE gel (4-15%, Bio-Rad). After the transfer, the blots were incubated overnight
at 4°C with polyclonal antibody against GFAP (mouse, 1:1000, Millipore) or IL-1β (1:1000,
rabbit, Millipore). For loading control, the blots were probed with β-tubulin or GAPDH
antibody (mouse, respectively 1:2000 and 1:20000, Sigma).

Quantitative real-time PCR (qPCR)

Animals (n = 4 per group) were terminally anesthetized with isoflurane at 1, 2, and
3 h after morphine injection. DRGs (L4/L5) and spinal cord dorsal horn segments (L4-L5)
were rapidly dissected and total RNA from each animal was isolated using RNeasy Plus
Mini kit (Qiagen, Valencia, CA). One microgram of RNA was reverse transcribed (RT)
using Omniscript reverse transcriptase according to the protocol of the manufacturer
(Qiagen). The sequences for the forward and reverse primers of GFAP and GAPDH are
included in Table 1. SYBR-green qPCR analyses were performed using the Opticon real-time PCR Detection
System (Bio-Rad, Hercules, CA) as described previously [70]. Briefly, qPCR amplification reactions contained RT products, 7.5 μl of 2X iQSYBR-green
mix (Bio-Rad), 300 nM of forward and reverse primers completed with nanopure water
for a final volume of 15 μl. The thermal cycling conditions were: 3 min at 95°C for
the polymerase activation, 45 cycles of 10 s at 95°C for denaturation, and 30 s at
60°C for annealing and extension, followed by a DNA dissociation curve for the determination
of the amplicon specificity. Analyses were carried out in triplicates and included
the different primer efficiencies obtained by a standard curve method.

Statistical analyses

All results are presented as means ± SEM. Differences between means were tested for
significance using Mann-Whitney test, 1-way or 2-way ANOVA followed by Bonferroni
post hoc test. Significance was determined at a level of P < 0.05.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

TB, YCL, and ZZX performed biochemical and histochemical experiments; TL performed
behavioral testing; RRJ, TB, and TL designed the experiments; and RRJ and TB wrote
the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This study was supported by NIH grants DE17794, NS54932, NS67686 to RRJ. TB was supported
by fellowships (PBLAP3-123417 and PA00P3-134165) from Switzerland. YC was supported
by a fellowship from Taiwan National Science Council (97-2918-I-006-012).